Urinary incontinence and bladder disease present unmet medical needs due, in part, to the inability of current therapies to correct loss of smooth muscle function associated with these disorders. Loss of functional smooth muscle in urethra accounts for a high degree of incontinence and the bladder is itself a smooth muscle organ.
Tissue engineered grafts have been created using host bladder cells to overcome these limitations. However, the use of bladder smooth muscle (SM) cells entails limitations as well. Oberpenning et al. (1999) Nat. Biotech. 17:149. In addition to the tremendous time and materials required for their expansion, studies have shown that cells from pathologic bladders, including neurogenic bladders, retain their neurogenic features in vitro. Lin et al. (2004) J. Urol. 171:1348.
The search for an ideal cell source for tissue engineering of the lower urinary tract has remained elusive. Atala and colleagues ((2004) Am. J. Transp. 4 Suppl.6:58) reported the use of autologous bladder smooth muscle (SM) cells to augment the bladder wall, however their experiments were limited to normal animals and required extensive cell culture and expansion. Bulking therapies of the urethra for the treatment of stress urinary incontinence (SUI) have faced similar limitations. The use of chrondrocytes as a cell source for injection into the urethra was described by Atala et al. (1944) J. Urol. 152:641. Chrondrocytes, harvested from the ear and expanded in culture, were implanted into the urethra and subsequently transformed into a rubbery-hard cartilage structure that provided bulk to the urethra. Atala et al. (1994) supra. While the results were encouraging, the long-term continence rates obtained using chondrocyte injections were similar to those achieved using traditional bulking agents such as collagen, silicone, and others. See Balmforth et al. (2003) Urology 62:52 and Beyt et al. (2001) Neuroalo. Urodyn. 20:157. Other investigators sought to use unprocessed fat as a bulking agent for SUI on the basis that fat is readily available, easily harvested, and immuno-compatible. Trockman et al. (1995) Urol. Clin. North Am. 22:665. While initially promising, less than 30% of the injected fat remained in the urethra after 35 days due to fat necrosis and inflammation. Olson et al. (1998) Urology 52:915.
More recently, investigators have described skeletal muscle cells as a source of tissue for the treatment of SUI and bladder dysfunction. Stenzl et al. (2000) World J. Urol. 18:44; Strasser et al. (2004) Urologe A. 43:1237; Corcos et al. (1999) Urology 54:815; and Yokayama et al. (2001) Mol. Urol. 5:67. Studies using injections of skeletal muscle cells have shown that the cells can remain viable in the rat urethra for up to 30 days and for up to 6 months in the bladder of SCID mice. Yokoyama et al. (2000) World J. Urol. 18:56 and Yokoyama et al. (2001) J. Urol. 165:271. Injected cells express myotubules and fast myosin heavy chain in vivo, consistent with striated muscle differentiation. Yokoyama et al. (2001) J. Urol. 165:271. Investigators reported improved sphincter contractility and elevated leak point pressures in animal models, Cannon et al. (2003) Urology 62:958 and Lee et al. (2003) J. Pelvic Floor Dysfunction 14:31 as well as improved continence in human subjects after injection of autologous skeletal muscle cells and fibroblasts into the rhabdosphincter and urethra. Strasser et al. (2004) Urologe A 43:1237. While these studies are very exciting examples of the potential of autologous cell transplantation for tissue engineering, it is still undetermined if skeletal muscle progenitors can differentiate into functional smooth muscle (SM) cells for long term applications outside of skeletal muscle regeneration. Lee et al. (2003) J. Pelvic Floor Dysfunction 14:31 and Timmermans et al. (2003) Cardiology 100:176. Moreover, there is a clear difference between smooth muscle and skeletal muscle cells.
Three kinds of muscle are found in all vertebrates: cardiac, smooth and skeletal muscle. Skeletal muscle is also called striated muscle and is usually attached to the skeleton. Its contraction is under voluntary control. Skeletal muscle is made up of thousands of cylindrical muscle fibers. Each one of these fibers contains an array of myofibrils, mitochondria, and endoplasmic reticulum and multiple nuclei. Thus skeletal muscle cells are multinucleated. Each myofibril is made of parallel filaments. The thick filaments are composed of the protein myosin and the thin filaments of troponin and tropomyosin. The contraction of skeletal muscle is controlled by the nervous system. In this respect, it differs from smooth muscle which can contract without stimulation from the nervous system. On the other hand, smooth muscle is found on the wall of the hollow organs of the body. Its contraction reduces the size of these structures. Thus it regulates the flow of blood in the arteries, moves food boluses through the intestinal tract, expels urine from the urinary bladder, is involved in uterine contractions and regulates the flow of air though the lungs among other functions. Unlike skeletal muscle, smooth muscle is made of single, spindle-shaped cells. The cell contains myosin and actin filaments that slide against each other to produce contraction of the cell. Unlike skeletal muscle, they do not depend on motor neurons to contract. However, neurons that reach smooth muscle can stimulate it by inducing contraction or relaxation. In addition, smooth muscle cells can respond to paracrine and hormonal stimulation. The contraction of smooth muscle tends to be slower than that of striated muscle and sustained for long period of time. Therefore, smooth muscle is capable of baseline tone.
Stem cells on the other hand, possess self-renewing capacity, long-term viability, and multilineage potential to overcome these limitations. Zuk (2001) supra. There are two general categories of stem cells—embryonic and adult stem cells. While the clinical potential of embryonic stem cells is enormous, their use is highly limited due to ethical considerations and cell regulation issues. As a result, much of the tissue engineering focus on stem cells has turned to the use of autologous adult mesenchymal stem cells. Bone marrow is the most studied source of mesenchymal progenitor cells. Bone marrow stem cells have been shown to differentiate into multiple cell types in vitro and in vivo. Hauner et al. (1987) J. Clin. Endocrinol. Metabol. 64(4):832. However, their utility for tissue engineering has been limited, due in part to bone marrow harvest morbidity, low cell yields, and patient specific serum requirements.
Compositions containing adipose-derived stem cells (ADSC) have previously been used for tissue construction, but never to reconstitute tissue containing functional smooth muscle. For example, WO 00/53795 and related applications disclose adipose-derived stem cells and lattices substantially free of adipocytes and red blood cells and clonal populations of connective tissue stem cells. The cells are disclosed to be useful alone or within biologically-compatible compositions, to generate differentiated tissues and structures in vivo and in vitro. U.S. Pat. No. 6,391,297 discloses a composition of an isolated human adipose tissue-derived stromal cell that has been differentiated to exhibit at least one characteristic of an osteoblast that can be used in vivo to repair bone and treat bone diseases. This adipose-derived osteoblast-like cell can be optionally genetically modified or combined with a matrix. U.S. Pat. No. 6,426,222 discloses methods for inducing osteoblast differentiation from human extramedullary adipose tissue by incubating the adipose tissue cells in a liquid nutrient medium that must contain a glucocorticoid.
U.S. Pat. Nos. 6,153,432 and 6,429,013 and U.S. Patent Application Publication Nos. 2004/0166096A1 and 2002/011564A1 also disclose the use of ADSCs for cartilage repair.
U.S. Patent Application Publ. Nos. 2005/0076396 and 2001/0033834 as well as PCT Publication WO 01/62901 disclose isolated adipose tissue-derived stromal cells that have been induced to express at least one phenotypic characteristic of a neuronal, astroglial, hematopoietic progenitor or hepatic cell.